1. Field of the Invention
The present invention is related to saccharides, including mono-, oligo-, and polysaccharides and methods for their analysis. More particularly, the present invention relates to fluorescent labelled mono- and polysaccharides and electrophoretic methods for their analysis. Advantageously, the present invention provides for the analysis and detection of picomole quantities of saccharides.
2. Description of Relevant Art
The analytical separation and detection of carbohydrates, simple sugars and oligo- and polysaccharides have been the subject of developmental efforts for decades. Traditional methods for separating and detecting these compounds have relied upon chromatographic methods, e.g. gas chromatography and high pressure liquid chromatography, wet chemical methods, and slab gel electrophoresis. Because carbohydrates lack outstanding structural features, the detection of carbohydrates at extremely low detection limits has been difficult. Carbohydrates absorb in the ultraviolet spectrum at 185 nm but their molar absorptivities are low. Amperometric detection is more sensitive than uv detection but suffers in that it is nonspecific and will detect a wide variety of compounds including amines and alcohols.
The more recent focus in biological research on the role of glycoproteins and carbohydrates in the structure and function of living cells has lead to an increase in the effort to detect carbohydrates, sugars, and oligosaccharides in trace quantities. Mono and polysaccharide analytical applications in which very low detection limits are desirable include carbohydrate and glycoprotein sequencing methods, the discovery and identification of new carbohydrates and drug development. Capillary electrophoresis in combination with amperometric detection has yielded detection of mono and oligosaccharides at concentration in the micromolar range. However, much lower detection limits are a goal of many analytical chemists and researchers.
More recently, electrophoresis has become an analytical tool of choice for smaller quantities of saccharides. The introduction of fluorescent detection in combination with slab gel and capillary electrophoresis has enhanced the detectability of carbohydrates and saccharides. For example, researchers have labelled mono- and oligosaccharides with fluorescent compounds and then electrophoretically separated and detected the labelled mono- and oligosaccharides. One of the earliest applications of fluorescent labelled sugars in slab gel electrophoresis was the reductive amination of sugars with 2-aminopyridine (AP). The AP reacts with the reducing end of mono- and polysaccharides. When the saccharide vicinal hydroxyls are complexed with borate at high pH (&gt;10), or the amine functionalities are protonated at low pH &lt;2.5), to form a charged compound, the labelled saccharide can be electrophoretically separated and detected with a fluorescent detector.
Another approach to the separation and detection of saccharides involves the reductive amination of monosaccharides and oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) followed by their electrophoretic separation and detection utilizing a CCD fluorescence imaging device. ANTS labelled oligosaccharides have been separated and detected also using capillary electrophoresis techniques. Other reagents which fluoresce and react with the reducing ends of saccharides or aminated saccharides include 5-aminonaphthalene-2-sulfonate (ANA) 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehdye (CBQCA), 4-aminobenzonitrile (ABN).
The above described fluorescing reagents used in combination with common fluorescent detectors and capillary electrophoresis offer enhanced detectability of mono- and polysaccharides. Notwithstanding the improvement in saccharide detection made possible by these reagents and methods, there is a continuing need for analytical techniques which provide for even lower detection limits for carbohydrates, monosaccharides, oligosaccharides, and polysaccharides.